Mutated arylsulfatase a

ABSTRACT

The present invention pertains to a novel treatment of pathologies caused by an increased synthesis or accumulation of sulfolipids such as sulfatide. The invention provides mutated arylsulfatase A (ARSA or ASA, EC 3.1.6.8) enzymes with increased activity towards sulfatide metabolization. The invention provides nucleic acids encoding the mutant ARSA, the use of the proteins and nucleic acids, as well as pharmaceutical compositions comprising them, in the treatment of lysosomal storage disorders (LSDs) such as metachromatic leukodystrophy (MLD).

FIELD OF THE INVENTION

The present invention pertains to a novel treatment of pathologiescaused by an increased synthesis or accumulation of sulfolipids such assulfatide. The invention provides mutated arylsulfatase A (ARSA or ASA,EC 3.1.6.8) enzymes with increased activity towards sulfatidemetabolization. The invention provides nucleic acids encoding the mutantARSA, the use of the proteins and nucleic acids, as well aspharmaceutical compositions comprising them, in the treatment oflysosomal storage disorders (LSDs) such as metachromatic leukodystrophy(MLD).

DESCRIPTION

Metachromatic leukodystrophy (MLD) (from the greek word leukos for“white”, dys for “lack of”, and troph for “growth”) is an autosomalrecessive lysosomal disorder caused by the deficiency in the enzymaticactivity of arylsulfatase A (ARSA or ASA, EC 3.1.6.8), resulting inimpaired degradation of 3-O-sulfogalactosylceramide (sulfatide), anessential sphingolipid of myelin (Gieselmann V & Krägeloh-Mann I,Neuropediatrics. 2010, 41, 1-6; Eckhardt M, Mol Neurobiol. 2008, 37:93-103.). ARSA hydrolyzes sulfatide to galactosylceramide and sulfateand is, due to the lack of alternative degradation pathways, essentialfor sulfatide recycling. Impairment of ARSA function results inincreased accumulation of sulfatide which clinically manifests inprogressive demyelination and neurological symptoms resulting in severedebilitation and eventually death of the affected patient. MLD is a raredisorder with a prevalence ranging from 1:40000 to 1:100000. Thedeficiency in the ARSA enzyme is caused by mutations in the ARSA gene inhomo- or heterozygosity encoding ARSA. Many mutations in the ARSA genehave been identified to date, but not all of these mutations cause thedeleterious MLD disease. MLD can manifest itself in young children(late-infantile form), where affected children typically begin showingsymptoms just after the first year of life (e.g., at about 15-24months), and death usually occurs about 5 years after onset of clinicalsymptoms. MLD can manifest itself in children (juvenile form), whereaffected children typically show cognitive impairment by about the ageof 3-10 years, and life-span can vary (e.g., in the range of 10-15 yearsafter onset of symptoms). MLD can manifest itself in adults at variousages beyond puberty (age 16 and later). The progression of suchadult-onset forms can vary greatly.

ARSA has been purified from a variety of sources including human liver,placenta, and urine. It is an acidic glycoprotein with a low isoelectricpoint. Above pH 6.5, the enzyme exists as a monomer with a molecularweight of approximately 60 kDa. ARSA undergoes a pH-dependentpolymerisation forming a dimer at pH 4.5. In human urine, the enzymeconsists of two non-identical subunits of 63 and 54 kDa. ARSA purifiedfrom human liver, placenta, and fibroblasts also consists of twosubunits of slightly different sizes varying between 55 and 64 kDa. Asin the case of other lysosomal enzymes, ARSA is synthesised onmembrane-bound ribosomes as a glycosylated precursor. It then passesthrough the endoplasmic reticulum and Golgi, where its N-linkedoligosaccharides are processed with the formation of phosphorylatedmannosyl residues that are required for lysosomal targeting via mannose6-phosphate receptor binding (Sommerlade et al., J Biol Chem. 1994, 269:20977-81; Coutinho M F et al., Mol genet metabol. 2012, 105: 542-550).

An unusual protein modification is essential for the enzymatic activityof all 17 human sulfatases known to date. It has been initiallyidentified in ARSA, arylsulfatase B (ARSB) and a sulfatase from thegreen alga Volvox carteri (Schmidt B et al. Cell. 1995, 82, 271-278,Selmer T et al. Eur J Biochem. 1996, 238, 341-345). This modificationleads to the conversion of an active site cysteine residue, which isconserved among the known sulfatases, into a 2-amino-3-oxopropionic acidresidue also termed Cα-formylglycine (FGly) (Schmidt B et al. Cell.1995, 82, 271-278). The formylglycine-generating enzyme (FGE) catalyzesthis conversion. A lack of FGE activity causes a combined functionaldeficiency of all human sulfatases, a severe lysosomal storage diseasecalled multiple sulfatase deficiency (MSD). In ARSA and ARSB theconversion of the Cys-69 and Cys-91 residue, respectively, to FGly isrequired for generating a catalytically active enzyme. Cys-69 isreferred to the precursor ARSA which has an 18 residue signal peptide.In the mature ARSA the mentioned cysteine residue is Cys-51. Furtherinvestigations have shown that a linear sequence of 16 residuessurrounding the Cys-51 in the mature ARSA is sufficient to direct theconversion and that the protein modification occurs after or at a latestage of co-translational protein translocation into the endoplasmicreticulum when the polypeptide is not yet folded to its native structure(Dierks T et al. Proc Natl Acad Sci. 1997, 94, 11963-1196).

Since MLD is caused by defective ARSA, most therapeutic approaches havetried to correct the biochemical defect by providing wild-type ARSA. Thedifferent methods and sources of wild-type ARSA constitute distincttherapeutic approaches (Sevin et al., J Inherit Metab Dis. 2007, 30,175-83). Hematopoietic stem cell transplantation (HSCT) is thetransplantation of hematopoietic stem cells from a healthy donor. Afterengraftment, progenies of donor-derived cells differentiate into thedifferent cell types of the hematopoeitic system and provide wild-typeARSA to patient's cells via a mannose 6-phosphate-dependentrelease-recapture pathway. This pathway is based on the pecularities ofthe sorting process of newly synthesized soluble lysosomal enzymes whichmay involve partial secretion of newly synthesized lysosomal enzymes andsubsequent uptake by neighbouring cells expressing mannose 6-phosphatereceptors on the cell surface. Many MLD patients have been treated byallogeneic HSCT with varying success. Enzyme replacement therapy (ERT)relies on providing recombinantly expressed wild-type human ARSA topatients. Repeated intravenous injection of therapeutic enzyme proved tobe effective in a number of lysosomal storage diseases and is clinicallyapproved for eight of them. For MLD, two clinical trials using eitherintravenous or intrathecal infusion of recombinant ARSA have beenlaunched (see below). Also gene therapy approaches are presently in theclinical evaluation. They are generally based on the overexpression ofwild-type ARSA in patient's own cells by transducing them withappropriate expression vectors. This can be done either by injectingappropriate expression vectors directly into the tissue (in vivo genetherapy) or by transducing patient's cells outside the body (ex vivogene therapy). Also in this treatment regimen the overexpressing cellsmay serve as an enzyme source for deficient cells. An ex vivo genetherapy approach using lentiviral gene transfer to overexpress ARSA inautologous CD34⁺ hematopoeitic stem cells is in a phase 1/2 clinicaltrial (see https://clinicaltrials.gov/ct2/show/NCT01560182). Theapproach was successful in a mouse model of MLD (Biffi A, et al., J ClinInvest. 2004, 113: 1118-29.). In another gene therapy trial (presentlyrecruiting patients) an adenovirus-associated vector encoding wild-typehuman ARSA will be injected directly into the brain of children affectedwith early on-set forms of MLD (seehttps://clinicaltrials.gov/ct2/show/NCT01801709). Also this in vivo genetherapy approach has demonstrated therapeutic benefit in a mouse modelof MLD (Piguet F et al. Hum Gene Ther. 2012, 23, 903-14). Other cellbased gene therapies for replacing ARSA try to use microencapsulatedrecombinant cells, oligodendrocyte progenitor cells, and neuralprogenitor cells as well as embryonic stem cells. All treatmentapproaches have limitations and bear certain risks. Also producing theenzyme for ERT in high purity and in large scale recombinantly has beena problem. Recently a study of ERT with wild-type ARSA was shown to beeffective in MLD (Dali et al., 2016, Mol Gen Metabol. 117, 73). Threecohorts of 6 patients each were treated with 10, 30 or 100 mg ofwild-type ARSA every two weeks in a total of 40 weeks treatmentschedule. To circumvent the blood-brain barrier, the enzyme wasadministered into the cerebrospinal fluid via intrathecal injections.Only few immunological adverse effects were observed. Although thisphase 1/2 clinical trial did not involve a placebo-treated controlgroup, conclusions can be drawn by comparing the different dose groups.Importantly, the group treated with 100 mg showed a significantlyreduced deterioration of motor functions compared to the group treatedwith 10 mg. However, still treatment effectivity suffers from targetingsufficient enzyme activity to the central nervous system. This isparticularily problematic if intravenous injection is used to provideenzyme to the patient as the blood-brain barrier prevents efficienttransfer of ARSA from the blood circulation to the brain parenchyma.Preclinical studies in mouse models of MLD had shown that weekly ARSAdoses of at least 20 mg per kg body weight are required to improvesulfatide storage in the brain (Matzner et al., Mol Ther, 2009, 17,600-606). The requirement of high doses in mice explains the failure ofa recent clinical trial testing repeated intravenous injection of up to5 mg/kg ARSA in early-onset MLD (seehttps://clinicaltrials.gov/ct2/show/results/NCT00418561). The enzymeactivity accumulating in the brain might have been below the thresholdrequired for therapeutic effects. Increasing the ARSA-doses is not apreferred solution to increase enzyme levels in the brain and treatmenteffectivity. Higher enzyme doses are more likely to induce thegeneration of neutralizing antibodies directed to the expressed proteinwhich might result in severe adverse effects including anaphylaxis.Therefore, there is a need to increase treatment effectivity of ARSAenzyme replacement. The same holds true for gene therapy because arelatively small number of producer cells has to supply ARSA activity toa large number of ARSA-deficient brain cells. In such approaches, anexcessive expression of wild-type ARSA might have adverse effectsbecause the overexpressed enzyme can deplete FGE from the endoplasmicreticulum of the producer cells and cause an inefficientpost-translational activation of ARSA and other cellular sulfatases.Also the cellular machinery generating mannose 6-phosphate residuesmight be overloaded, resulting in the delivery of uptake-incompetentenzyme.

The above problems of ERT and gene therapy are solved in a first aspectby providing a mutated ARSA enzyme, or a functional fragment thereof,having increased enzymatic activity compared to the wild-type sequence.The invention therefore pertains in preferred embodiments to a mutatedarylsulfatase A (ARSA) enzyme, comprising an amino acid sequence with atleast 80%, 85%, 90%, 95%, 96%, 97%, preferably at least 85% or 90% mostpreferably at least 99% sequence identity to SEQ ID NO: 1 (human ARSAenzyme), wherein the amino acid sequence of the mutated ARSA enzyme, orthe functional fragment thereof, when aligned to the sequence of SEQ IDNO: 1, comprises at least one mutation compared to the sequence betweenresidues 100 and 400 of SEQ ID NO: 1.

As used herein, the terms “identical” or percent “identity”, when usedanywhere herein in the context of two or more nucleic acid orprotein/polypeptide sequences, refer to two or more sequences orsubsequences that are the same or have (or have at least) a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., at, or at least, about 60% identity, preferably at, or at least,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94%, identity, and morepreferably at, or at least, about 95%, 96%, 97%, 98%, 99%, or higheridentity over a specified region—preferably over their full lengthsequences—, when compared and aligned for maximum correspondence overthe comparison window or designated region) as measured using a sequencecomparison algorithms, or by manual alignment and visual inspection(see, e.g., NCBI web site). In a particular embodiment, for example whencomparing the protein or nucleic acid sequence of a mutated ARSA withwild-type ARSA, the percentage identity can be determined by the Blastsearches or local alignments; in particular for amino acid identity,those using BLASTP 2.2.28+ with the following parameters: Matrix:BLOSUM62; Gap Penalties: Existence: 11, Extension: 1; Neighboring wordsthreshold: 11; Window for multiple hits: 40.

The term “mutation” refers to, in the context of a polynucleotide, amodification to the polynucleotide sequence resulting in a change in thesequence of a polynucleotide with reference to a precursorpolynucleotide sequence. A mutant polynucleotide sequence can refer toan alteration that does not change the encoded amino acid sequence, forexample, with regard to codon optimization for expression purposes, orthat modifies a codon in such a way as to result in a modification ofthe encoded amino acid sequence. Mutations can be introduced into apolynucleotide through any number of methods known to those of ordinaryskill in the art, including random mutagenesis, site-specificmutagenesis, oligonucleotide directed mutagenesis, gene shuffling,directed evolution techniques, combinatorial mutagenesis, sitesaturation mutagenesis among others.

“Mutation” or “mutated” means, in the context of a protein, amodification to the amino acid sequence resulting in a change in thesequence of a protein with reference to a precursor protein sequence. Amutation can refer to a substitution of one amino acid with anotheramino acid, an insertion or a deletion of one or more amino acidresidues. Specifically, a mutation can also be the replacement of anamino acid with a non-natural amino acid, or with a chemically-modifiedamino acid or like residues. A mutation can also be a truncation (e.g.,a deletion or interruption) in a sequence or a subsequence from theprecursor sequence. A mutation may also be an addition of a subsequence(e.g., two or more amino acids in a stretch, which are inserted betweentwo contiguous amino acids in a precursor protein sequence) within aprotein, or at either terminal end of a protein, thereby increasing thelength of (or elongating) the protein. A mutation can be made bymodifying the DNA sequence corresponding to the precursor protein.Mutations can be introduced into a protein sequence by known methods inthe art, for example, by creating synthetic DNA sequences that encodethe mutation with reference to precursor proteins, or chemicallyaltering the protein itself. A “mutant” as used herein is a proteincomprising a mutation. For example, it is also possible to make a mutantby replacing a portion of ARSA with a wild-type sequence thatcorresponds to such portion but includes a desired variation at aspecific position that is naturally-occurring in the wild-type sequence.

The use of the mutated ARSA enzymes, or of the functional fragmentthereof, of the present invention overcomes the problems in the artbecause their increased catalytic activity to metabolize sulfatidesallows to maintain low enzyme concentrations/expressions whileincreasing enzyme activity. Also, problems of expressing sufficientamount of enzyme activity either recombinantly (ERT) or in situ (genetherapy) is overcome by the herein provided highly active mutated ARSAvariant. The mutated ARSA of the invention shows a up to 5 foldincreased activity compared to the human wild-type enzyme.

In preferred embodiments of the invention, the mutated ARSA enzyme aminoacid sequence, or of the functional fragment thereof, when aligned tothe sequence of SEQ ID NO: 1, comprises at least one mutation comparedto the sequence between residues 150 and 350 of SEQ ID NO: 1. Morepreferably the at least one mutation in the mutated ARSA of theinvention is located between residues 180 to 220, and/or 260to 320 ofSEQ ID NO: 1, respectively their corresponding amino acid positions inthe mutated sequence. A mutated ARSA is preferred, wherein the aminoacid sequence when aligned to the sequence of SEQ ID NO: 1, comprises atleast one mutation compared to the sequence between residues 195 to 210,and/or 280 to 300 of SEQ ID NO: 1. In other embodiments the mutated ARSAenzyme amino acid sequence, or of the functional fragment thereof, whenaligned to the sequence of SEQ ID NO: 1, comprises at least one mutationat amino acid positions 202, 286 and/or 291 of SEQ ID NO: 1.

A preferred mutated ARSA of the invention is a protein having at least65%, 70 %, 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94%, identity, and morepreferably at, or at least, about 95%, 96%, 97%, 98%, 99% or 100%sequence identity compared to the sequence shown in SEQ ID NO: 3 or 4.Also included are functional fragments of these proteins that retain theARSA catalytic activity. Preferably, however, such mutated ARSA of theinvention, or their functional fragments, comprise at least one aminoacid mutation at positions corresponding to amino acids 202, 286 and/or291 of SEQ ID NO: 1.

Therefore also provided in some embodiments is a functional fragment ofa mutated ARSA of the invention. The functional fragment preferablycomprises, or consists of, or consists essentially of, 50 amino acids,preferably 80, more preferably at least 100, more preferably 200, 300 or400 or 450 amino acids, under the provision that said functionalfragment of a mutated ARSA retains the ARSA catalytic activity asdescribed herein, and preferably comprises at least one amino acidmutation at positions corresponding to amino acids 202, 286 and/or 291of SEQ ID NO: 1.

As mentioned the mutation introduced into the ARSA according to theinvention is preferably selected from a substitution, deletion,addition, insertion or amino acid modification, and preferably is anamino acid substitution. Most preferably, the mutated sequence of themutated ARSA of the invention constitutes a murinization of the humanARSA amino acid sequence. A “murinization” or “murinizing” in context ofthe present invention shall be understood to refer to the introductionof a murine ARSA amino acid or nucleic acid sequence into the amino acidor nucleic acid sequence of a homologous non-murine ARSA protein orgene—preferably human ARSA. Therefore, as an example, a human ARSAsequence is considered to be “murinized”, if into the human sequence atat least one position the amino acid sequence of the correspondingmurine wild type enzyme is introduced. Murinization may include theexchange of only one amino acid from non-mouse to mouse, or of multipleamino acids.

In some embodiments of the invention, the mutated ARSA enzyme amino acidsequence, or of the functional fragment thereof, when aligned to thesequence of SEQ ID NO: 1, comprises at least one mutation selected fromM202V, T286L and/or R291N compared to SEQ ID NO: 1, preferably of atleast M202V. In other embodiments any one of amino acid substitutionsM202V, T286L and/or R291N, may be accompanied by one or more additionalamino acid mutations. In other embodiments a mutated ARSA is preferredwherein the mutated ARSA enzyme amino acid sequence, or of thefunctional fragment thereof, when aligned to the sequence of SEQ ID NO:1, comprises the mutations selected from the group consisting of M202V,T286L and R291N compared to SEQ ID NO: 1.

A mutated ARSA is preferred wherein the mutated ARSA enzyme amino acidsequence, or of the functional fragment thereof, when aligned to thesequence of SEQ ID NO: 1, comprises at least two mutations, preferablyall three, selected from M202V, T286L and/or R291N of SEQ ID NO: 1,preferably of at least M202V.

The mutated ARSA enzyme, or the functional fragment thereof, of theinvention in preferred embodiments retains an enzymatic activity ofdegradation of sulfatides, preferably an activity of degradation of3-O-sulfogalactosylceramide into galactosylceramide and sulfate.Preferably the mutated ARSA enzyme, or the functional fragment thereof,of the invention has an increased aforementioned activity compared tohuman wild-type ARSA.

The mutated ARSA of the invention is in preferred embodiments anisolated ARSA or a recombinant ARSA polypeptide. The term “recombinant”or “recombinantly produced” in context of the invention means that aprotein or peptide is expressed via an artificially introduced exogenousnucleic acid sequence in a biological cell. Recombinant expression isusually performed by using expression vectors as described hereinelsewhere.

In another aspect the problem is solved by an isolated nucleic acidcomprising a sequence coding for the mutated ARSA enzyme as describedherein before, or for a functional fragment of a mutated ARSA enzyme asdescribed herein before. The term “encoding” or more simply “coding”refers to the ability of a nucleotide sequence to code for one or moreamino acids. The term does not require a start or stop codon. An aminoacid sequence can be encoded in any one of six different reading framesprovided by a polynucleotide sequence and its complement. An amino acidsequence can be encoded by desoxyribonucleic acid (DNA), ribonucleicacid (RNA), or artificially synthesized polymers similar to DNA or RNA.

Another aspect of the invention provides a vector, comprising thenucleic acid of the invention. A “vector” may be any agent that is ableto deliver or maintain a nucleic acid in a host cell and includes, forexample, but is not limited to, plasmids (e.g., DNA plasmids), nakednucleic acids, viral vectors, viruses, nucleic acids complexed with oneor more polypeptide or other molecules, as well as nucleic acidsimmobilized onto solid phase particles. Vectors are described in detailbelow. A vector can be useful as an agent for delivering or maintainingan exogenous gene and/or protein in a host cell. A vector may be capableof transducing, transfecting, or transforming a cell, thereby causingthe cell to replicate or express nucleic acids and/or proteins otherthan those native to the cell or in a manner not native to the cell. Thetarget cell may be a cell maintained under cell culture conditions or inother in vivo embodiments, being part of a living organism. A vector mayinclude materials to aid in achieving entry of a nucleic acid into thecell, such as a viral particle, liposome, protein coating, or the like.Any method of transferring a nucleic acid into the cell may be used;unless otherwise indicated, the term vector does not imply anyparticular method of delivering a nucleic acid into a cell or imply thatany particular cell type is the subject of transduction. The presentinvention is not limited to any specific vector for delivery ormaintenance of any nucleic acid of the invention, including, e.g., anucleic acid encoding a mutant ARSA polypeptide of the invention or afragment thereof.

Preferably the vector of the invention is an expression vector. The term“expression vector” typically refers to a nucleic acid construct orsequence, generated recombinantly or synthetically, with a series ofspecific nucleic acid elements that permit transcription of a particularnucleic acid in a host cell. The expression vector typically includes anucleic acid to be transcribed—the mutated ARSA of theinvention—operably linked to a promoter. The term “expression” includesany step involved in the production of the polypeptide including, butnot limited to, transcription, post-transcriptional modification,translation, post-translational modification, and/or secretion. Apreferred vector of the invention is a plant-specific, bacterial, yeast,insect, vertebrate, preferably mammalian, or a viral vector, preferablyretroviral and adeno-associated viral vector. Preferred vectors of theinvention are suitable for use in gene therapy, preferably gene therapybased on transformation of autologous adult stem cells.

In another aspect there is also provided a recombinant cell comprising amutated ARSA enzyme, or the functional fragment thereof, a nucleic acid,or a vector or expression vector of the invention as described herein. A“recombinant cell” or also referred to as “host cell” is any cell thatis susceptible to transformation with a nucleic acid. Preferably therecombinant or host cell of the invention is a plant cell, bacterialcell, yeast cell, an insect cell or a vertebrate, preferably amammalian, cell. A preferred recombinant cell is selected from a cellsuitable for recombinant expression of the mutated ARSA of theinvention. Most preferred is a Chinese hamster ovary (CHO) cell. Alsopreferred are human cells, preferably autologous human cells derivedfrom patient suffering from a disease described herein that is treatablewith a mutated ARSA of the invention. A preferred human cell is ahematopoietic stem cell (HSC).

In another aspect there is provided a pharmaceutical compositioncomprising a mutated ARSA enzyme, or the functional fragment thereof, anucleic acid, a vector, or a recombinant cell of the invention asdescribed before, together with a pharmaceutically acceptable carrier,stabilizer and/or excipient.

In the following the mutated ARSA, nucleic acids encoding the same,vectors and cells comprising these nucleic acids or mutated proteins, aswell as pharmaceutical compositions thereof, will be referred togenerally as “compounds of the invention”.

As used herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, solubilizers, fillers,stabilizers, binders, absorbents, bases, buffering agents, lubricants,controlled release vehicles, diluents, emulsifying agents, humectants,lubricants, dispersion media, coatings, antibacterial or antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well-known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary agents can also be incorporated into the compositions. Incertain embodiments, the pharmaceutically acceptable carrier comprisesserum albumin.

The pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intrathecal,intracerebroventricular, intraparenchymal, intra-arterial, intravenous,intradermal, subcutaneous, oral, transdermal (topical) and transmucosaladministration. The term “intrathecal,” as used herein, means introducedinto or occurring in the space under the arachnoid membrane which coversthe brain and spinal cord. The term “intracerebroventricular” refers toadministration of a composition into the ventricular system of thebrain, e.g., via injection, infusion, or implantation (for example, intoa ventricle of the brain). As used herein, the term “intraparenchymal”can refer to an administration directly to brain tissue. In otherinstances, intraparenchymal administration may be directed to any brainregion where delivery of one or more compounds of the invention iseffective to mitigate or prevent one or more of disorders as describedherein.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine; propylene glycol or other syntheticsolvents; anti-bacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride, mannitol or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the injectable composition should be sterile and should be fluidto the extent that easy syringability exists. It must be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, mannitol,propylene glycol, and liquid polyetheylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a compound of the invention such as a mutated ARSA) inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound of the invention into a sterile vehicle which contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orStertes; a glidant such as colloidal silicon dioxide; a sweetening agentsuch as sucrose or saccharin; or a flavoring agent such as peppermint,methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the pharmaceutical compositions areformulated into ointments, salves, gels, or creams as generally known inthe art.

In certain embodiments, the pharmaceutical composition is formulated forsustained or con-trolled release of the active ingredient.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen, serumalbumin, polyorthoesters, polylactic acid, poly(butyl cyanoacrylate),and poly(lactic-coglycolic) acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from e.g. Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein includesphysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of ad-ministration utilized. For any compoundused in the method of the invention, the therapeutically effective dosecan be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. The pharmaceuticalcompositions can be included in a container, pack, or dispenser togetherwith instructions for administration.

The problem is furthermore solved by a medical use of the compounds ofthe invention in the treatment of a disease. The disease is preferably adisease characterized by a pathological enzymatic insufficiency ofendogenous ARSA. Generally preferred diseases are demyelinatingdisorders. In other preferred embodiments the disease is aleukodystrophy. A leukodystrophy in context with the present inventionis preferably selected from metachromatic leukodystrophy, multiplesulfatase deficiency, Krabbe disease, adrenoleukodystrophy,Pelizaeus-Merzbacher disease, Canavan disease, Childhood Ataxia withCentral Hypomyelination or CACH (also known as Vanishing White MatterDisease), Alexander disease, Refsum disease, and cerebrotendinousxanthomatosis. In most preferred embodiments of the invention thedisease is metachromatic leukodystrophy (MLD).

Compositions and methods of the present invention may be used toeffectively treat individuals suffering from or susceptible to MLD. Theterms, “treat” or “treatment”, as used herein, refers to amelioration ofone or more symptoms associated with the disease, prevention or delay ofthe onset of one or more symptoms of the disease, and/or lessening ofthe severity or frequency of one or more symptoms of the disease.Exemplary symptoms include, but are not limited to, intracranialpressure, hydrocephalus ex vacua, accumulated sulfated glycolipids inthe myelin sheaths in the central and peripheral nervous system and invisceral organs, progressive demyelination and axonal loss within theCNS and PNS, and/or motor and cognitive dysfunction, like gaitdisturbances, mental regression, ataxia, loss of speech, spastictetraparesis, or optic atrophy.

In some embodiments, treatment refers to partially or completealleviation, amelioration, relief, inhibition, delaying onset, reducingseverity and/or incidence of neurological impairment in an MLD patient.As used herein, the term “neurological impairment” includes varioussymptoms associated with impairment of the central nervous system (brainand spinal cord). In some embodiments, various symptoms of MLD areassociated with impairment of the peripheral nervous system (PNS). Insome embodiments, neurological impairment in an MLD patient ischaracterized by decline in gross motor function. It will be appreciatedthat gross motor function may be assessed by any appropriate methodknown to the skilled artisan.

In some embodiments, treatment refers to decreased sulfatideaccumulation in various tissues. In some embodiments, treatment refersto decreased sulfatide accumulation in brain target tissues, spinal cordneurons, and/or peripheral target tissues. In certain embodiments,sulfatide accumulation is decreased by about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,100% or more as compared to a control. In some embodiments, sulfatideaccumulation is decreased by at least 1-fold, 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold as compared to acontrol. It will be appreciated that sulfatide storage may be assessedby any appropriate method. For example, in some embodiments, sulfatidestorage is measured by alcian blue staining. In some embodiments,sulfatide storage is measured by high-performance liquid chromatography,thin layer chromatography or mass spectrometry.

In some embodiments, treatment refers to reduced vacuolization or areduced number and/or size of alcian blue-positive storage deposits inneurons (e.g. in nuclei of the medulla oblongata and pons, and inseveral nuclei of midbrain and forebrain,), astrocytes, oligodendroctes,Schwann cells and/or microglial cells. In certain embodiments,vacuolization or storage deposits in these cell types are decreased byabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 100% or more as compared to a control. Insome embodiments, vacuolization or storage deposits are decreased by atleast 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold or 10-fold as compared to a control.

In some embodiments, treatment refers to increased ARSA enzyme activityin various tissues. In some embodiments, treatment refers to increasedARSA enzyme activity in brain target tissues, spinal cord, peripheralnerves and/or other peripheral target tissues. ARSA enzyme activity canbe measured by using artificial substrates such as para-nitrocatecholsulfate and 4-methylumbelliferyl sulfate or by using the naturalsubstrate 3-O-sulfogalactosylceramide. In some embodiments, ARSA enzymeactivity is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%,400%, 500%, 600%, 700%, 800%, 900%, 1000% or more as compared to acontrol. In some embodiments, ARSA enzyme activity is increased by atleast 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold or 10-fold as compared to a control.

In some embodiments, increased ARSA enzymatic activity is at leastapproximately 10 nmol/hr/mg, 20 nmol/hr/mg, 40 nmol/hr/mg, 50nmol/hr/mg, 60 nmol/hr/mg, 70 nmol/hr/mg, 80 nmol/hr/mg, 90 nmol/hr/mg,100 nmol/hr/mg, 150 nmol/hr/mg, 200 nmol/hr/mg, 250 nmol/hr/mg, 300nmol/hr/mg, 350 nmol/hr/mg, 400 nmol/hr/mg, 450 nmol/hr/mg, 500nmol/hr/mg, 550 nmol/hr/mg, 600 nmol/hr/mg or more. In some embodiments,ARSA enzymatic activity is increased in the lumbar region. In someembodiments, increased ARSA enzymatic activity in the lumbar region isat least approximately 2000 nmol/hr/mg, 3000 nmol/hr/mg, 4000nmol/hr/mg, 5000 nmol/hr/mg, 6000 nmol/hr/mg, 7000 nmol/hr/mg, 8000nmol/hr/mg, 9000 nmol/hr/mg, 10,000 nmol/hr/mg, or more.

In some embodiments, treatment refers to decreased progression of lossof cognitive ability. In certain embodiments, progression of loss ofcognitive ability is decreased by about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% ormore as compared to a control. In some embodiments, treatment refers todecreased developmental delay. In certain embodiments, developmentaldelay is decreased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more ascompared to a control.

In some embodiments, treatment refers to increased survival (e.g.survival time). For example, treatment can result in an increased lifeexpectancy of a patient. In some embodiments, treatment according to thepresent invention results in an increased life expectancy of a patientby more than about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about95%, about 100%, about 105%, about 110%, about 115%, about 120%, about125%, about 130%, about 135%, about 140%, about 145%, about 150%, about155%, about 160%, about 165%, about 170%, about 175%, about 180%, about185%, about 190%, about 195%, about 200% or more, as compared to theaverage life expectancy of one or more control individuals with similardisease without treatment. In some embodiments, treatment according tothe present invention results in an increased life expectancy of apatient by more than about 6 month, about 7 months, about 8 months,about 9 months, about 10 months, about 11 months, about 12 months, about2 years, about 3 years, about 4 years, about 5 years, about 6 years,about 7 years, about 8 years, about 9 years, about 10 years or more, ascompared to the average life expectancy of one or more controlindividuals with similar disease without treatment. In some embodiments,treatment according to the present invention results in long termsurvival of a patient. As used herein, the term “long term survival”refers to a survival time or life expectancy longer than about 40 years,45 years, 50 years, 55 years, 60 years, or longer.

The terms, “improve,” “increase” or “reduce,” as used herein, indicatevalues that are relative to a control. In some embodiments, a suitablecontrol is a baseline measurement, such as a measurement in the sameindividual prior to initiation of the treatment described herein, or ameasurement in a control individual (or multiple control individuals) inthe absence of the treatment described herein. A “control individual” isan individual afflicted with the same form MLD (e.g., late-infantile,juvenile, or adult-onset form), who is about the same age and/or genderas the individual being treated (to ensure that the stages of thedisease in the treated individual and the control individual(s) arecomparable.

The individual (also referred to as “patient” or “subject”) beingtreated is an individual (fetus, infant, child, adolescent, or adulthuman) having MLD or having the potential to develop MLD. The individualcan have residual endogenous ARSA expression and/or activity, or nomeasurable activity. For example, the individual having MLD may haveARSA expression levels that are less than about 30-50%, less than about25-30%, less than about 20-25%, less than about 10-15%, less than about5-10%, less than about 0.1-5% of normal ARSA expression levels.

In some embodiments, the individual is an individual who has beenrecently diagnosed with the disease. Typically, early treatment(treatment commencing as soon as possible after diagnosis) is importantto minimize the effects of the disease and to maximize the benefits oftreatment.

A treatment according to the invention preferably comprises theadministration of a therapeutically effective amount of the compound ofthe invention to a subject in need of the treatment.

Preferred wherein the treatment comprises the intravenous,intracerebral, intrathecal and/or intracerebroventricular injection orinfusion of a therapeutically effective amount of the compound to asubject in need of the treatment.

The compounds of the invention for use in therapeutic treatments areadministered to a patient suffering from a disorder as mentioned herein,in therapeutically effective doses. As used herein, the term“therapeutically effective dose” intends that dose of ARSA that achievesa therapeutic effect, and is typically in the range of about 0.05 mg/kgto about 1.0 mg/kg/day for both children and adults, and more preferablyof about 0.075 mg/kg/day to about 0.3 mg/kg/day. The therapeutic dose ofcompound of the invention can be administered as a single dose ordivided doses given in certain intervals of time, for example as two,three, four or more daily doses. A preferred treatment comprises theadministration of 0.1 to 1000 mg of mutated ARSA enzyme, or thefunctional fragment thereof, of the invention to a subject in need ofthe treatment, for example once a week, once every two weeks, or onceevery three weeks, for at least 2, preferably 3, 4, 5, 6, 7, 8, 9, 10,11, or 12 months, or longer.

In some embodiments, the treatment of the invention is a gene therapy oran enzyme replacement therapy. The replacement enzyme suitable for theinvention is preferably a mutant ARSA as described herein before. Thereplacement enzyme suitable for the present invention may be produced byany available means. For example, replacement enzymes may berecombinantly produced by utilizing a host cell system engineered toexpress a replacement enzyme-encoding nucleic acid. Where enzymes arerecombinantly produced, any expression system can be used. To give but afew examples, known expression systems include, for example, egg,baculovirus, plant, yeast, or mammalian cells.

In some embodiments, mutated ARSA enzymes, or the functional fragmentsthereof, suitable for the present invention are produced in mammaliancells. Non-limiting examples of mammalian cells that may be used inaccordance with the present invention include BALB/c mouse myeloma line(NSO/1, ECACC No:85110503); human retinoblasts (PER.C6, CruCell, Leiden,The Netherlands); monkey kidney CV1 line transformed by SV40 (COS-7,ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subclonedfor growth in suspension culture, Graham et al, J. Gen Virol, 36:59,1977); human fibrosarcoma cell line (e.g., HT1080); baby hamster kidneycells (BHK, ATCC CCL 10); Chinese hamster ovary cells +/−DHFR (CHO,Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980); mouseSertoli cells (TM4, Mather, Biol. Reprod., 23:243-251, 1980); monkeykidney cells (CV1 ATCC CCL 70); African green monkey kidney cells(VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCCCCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells(BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCCCCL51); TRI cells (Mather et al, Annals N.Y. Acad. Sci., 383:44-68,1982); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

In some embodiments, the mutated ARSA enzymes, or the functionalfragments thereof, delivered using a method of the invention contain amoiety that binds to a receptor on the surface of brain cells tofacilitate cellular uptake and/or lysosomal targeting. For example, sucha receptor may be the cation—independent mannose-6-phosphate receptor(CI-MPR) which binds the mannose-6-phosphate (M6P) residues. Inaddition, the CI-MPR also binds other proteins including IGF-II. In someembodiments, a replacement enzyme suitable for the present inventioncontains M6P residues on the surface of the protein. In someembodiments, a replacement enzyme suitable for the present invention maycontain bis-phosphorylated oligosaccharides which have higher bindingaffinity to the CI-MPR. In some embodiments, a suitable enzyme containsup to about an average of about at least 20% bis-phosphorylatedoligosaccharides per enzyme. In other embodiments, a suitable enzyme maycontain about 10%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%bis-phosphorylated oligosaccharides per enzyme. While suchbis-phosphorylated oligosaccharides may be naturally present on theenzyme, it should be noted that the enzymes may be modified to possesssuch oligosaccharides. For example, suitable replacement enzymes may bemodified by certain enzymes which are capable of catalyzing the transferof N-acetylglucosaminei-phosphate from UDP-N-acetylglucosamine to the 6′position of alpha-1,2-linked mannoses on lysosomal enzymes. Methods andcompositions for producing and using such enzymes are described by, forexample, Canfield et al. in U.S. Pat. No. 6,537,785, and U.S. Pat. No.6,534,300, each incorporated herein by reference.

In some embodiments, mutated ARSA enyzmes for use in the presentinvention may be conjugated or fused to a lysosomal targeting moietythat is capable of binding to a receptor on the surface of brain cells.A suitable lysosomal targeting moiety can be IGF-I, IGF-II, RAP,apolipoprotein E, p97, and variants, homologues or fragments thereof(e.g., including those peptide having a sequence at least 70%, 75%, 80%,85%, 90%, or 95% identical to a wild-type mature human IGF-I, IGF-II,RAP, apolipoprotein E, p97 peptide sequence).

In some embodiments, a therapeutic protein includes a targeting moiety(e.g., a lysosome targeting sequence) and/or a membrane -penetratingpeptide. In some embodiments, a targeting sequence and/or amembrane-penetrating peptide is an intrinsic part of the therapeuticmoiety (e.g., via a chemical linkage, via a fusion protein). In someembodiments, a targeting sequence contains a mannose-6-phosphate moiety.In some embodiments, a targeting sequence contains an IGF-I moiety. Insome embodiments, a targeting sequence contains an IGF-II moiety.

A preferred treatment of a LSD of the invention involves gene therapy.Such methods may include the transformation of a human cell with amutated ARSA and infusion of the so produced cell into a patientaccording to the above described preferred routes. Preferably genetherapy may comprise obtaining autologous adult stem cells of a patient,preferably HSCs. These cells are in a next step genetically altered toexpress a mutated ARSA of the invention. Genetically alteration may beachieved by either transforming the cell with an expression vector ofthe invention, or alternatively, by directly mutating the HSC endogenousARSA using for example gene editing (e.g. CRISPR/Cas9 approaches). Ifthe endogenous ARSA comprises one or more mutations decreasing ARSAactivity and/or expression, the approach also comprises repairing ARSAdeficiency by reconstitution of the wild-type sequence at the respectivepositions. In general the present invention also pertains to methods forgenerating a mutated ARSA as described before, by providing a targetcell which endogenously expresses human ARSA, and introducing the ARSAmutations of the invention into the endogenous human ARSA sequence.

The pharmaceutical compositions according to the invention are inpreferred embodiments suitable for CNS delivery of the compounds of theinvention.

In another aspect there is also provided a method for producing thecompounds of the invention.

In another aspect the invention also pertains to a method for designingand/or producing a mutated ARSA enzyme, or a functional fragmentthereof, comprising the steps of

-   -   (a) providing a parent ARSA enzyme-encoding nucleic acid        sequence which encodes a parent ARSA enzyme having an amino acid        sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,        preferably 100% sequence identity to SEQ ID NO: 1,    -   (b) introducing into said parent ARSA enzyme-encoding nucleic        acid sequence at least one mutation, thereby generating a        mutated ARSA enzyme(or functional fragment)-encoding nucleic        acid sequence, wherein the mutated ARSA enzyme(or functional        fragment)-encoding nucleic acid sequence encodes a mutated ARSA        enzyme, or functional fragment thereof, comprising a mutated        ARSA enzyme amino acid sequence, that, when aligned to the        sequence of SEQ ID NO: 1, comprises at least one mutated amino        acid residue compared to the sequence of SEQ ID NO: 1 between        residues 100 and 400, and wherein said at least one mutated        amino acid residue constitutes a mutation when compared to the        amino acid sequence of the parent ARSA enzyme,    -   (c) Optionally, expressing said mutated ARSA enzyme, or        functional fragment thereof, encoding nucleic acid sequence, to        obtain a mutated ARSA enzyme, or the functional fragment        thereof.

In this aspect the mutated ARSA enzyme amino acid sequence, or aminoacid sequence of the functional fragment, when aligned to the sequenceof SEQ ID NO: 1, comprises preferably at least one mutation compared tothe sequence SEQ ID NO: 1 between residues 150 and 350, preferablybetween residues 180 to 220, and/or 260 to 320 of SEQ ID NO: 1, morepreferably between residues 195 to 210, and/or 280to 300 of SEQ ID NO:1, most preferably at amino acid positions 202, 286 and/or 291 of SEQ IDNO: 1.

The present invention will now be further described in the followingexamples with reference to the accompanying figures and sequences,nevertheless, without being limited thereto. For the purposes of thepresent invention, all references as cited herein are incorporated byreference in their entireties. In the Figures:

FIG. 1: Activity of murine and human ARSA towards its natural substratesulfatide (sulf).

FIG. 2: Alignment of the amino acid sequences of human and murine ARSA.Sequences are deduced from the cDNAs (Stein C et al., J Biol Chem.,1989, 264, 1252-9; Kreysing et al., Genomics., 1994, 19, 249-56.).Informations about functional and structural elements are from Lukatelaet al., Biochemistry, 1998, 37, 3654-64.

FIG. 3: Schematic representation of murinized ARSA constructs withsingle exchanges of human-specific mutations and variable domains. Blackarrows indicate regions where murine sequences were introduced.

FIG. 4: Illustration of the experimental procedures to generate andanalyse chimeric ARSA polypeptides.

FIG. 5: Murinization of individual variable domains. Black arrowsindicate regions where murine sequences were introduced.

FIG. 6: Murinization of groups of variable domains. Black arrowsindicate regions where murine sequences were introduced.

FIG. 7: Murinization of amino acids in the variable domains v4 and v6.Black arrows indicate regions where murine sequences were introduced.

FIG. 8: Murinization of M202 plus other amino acids in v4 or v6.

FIG. 9: Partial and complete murinization of v5 in the ARSA mutantM202V, T286L, R291N.

FIG. 10: Specific activities of the ARSA mutants M202V and M202V, T286L,R291N.

FIG. 11: Endocytosis of ARSA mutants by CHO-K1 cells. Data are based onactivity measurements as described in materials and methods. Bars showmeans±SDs of three independent feeding experiments. A industriallymanufactured wildtype human ARSA [hARSA (control)] obtained from Zymenex(HiHerød, Denmark) was used as a control (open bar).

FIG. 12: Stability of ARSA mutants. hASA—human ARSA, mASA—murine ARSA.(A) Shelf lives of the indicated ARSAs after incubation in Tris-bufferedsaline pH 7.4 for up to 10 days at 4° C. (B) Effect of repeatedfreeze-thaw cycles on enzyme activity. (C) Intracellular stability.CHO-K1 cells were fed for 24 h with recombinant ARSAs (2.5 μg/ml) asindicated. Then fresh medium was added and the CHO-K1 cells wereharvested after chase times of 0, 24, 48 and 72 h, respectively. ForWestern blotting a mixture of two polyclonal rabbit anti-ARSA antiserawas used to detect intracellular murine and human ARSA on the samefilter. Homogenates of CHO-K1 cells cultured without ARSA were used as anegative control (neg).

FIG. 13: Anti-ARSA antibodies in serum of humanized MLD mice treated byERT with different ARSA-variants. Antibody concentrations were measuredby immunoprecipitation in sera of 12 mice treated with either human ARSA(hARSA), ARSA_M202V, ARSA_M202V,T286L,R291N or murine ARSA (mARSA) (n=3mice per group). Two antisera from rabbits that had been immunized withwildtype human ARSA and two sera from mice that had been mock-treatedwith Tris-buffered saline were taken as positive (pos #1, #2) andnegative controls (neg #1, #2), respectively.

SEQ ID NO: 1 shows the sequence of wild type human ARSA proteinincluding the signal peptide (underlined) and most preferred positionsfor mutation (bold and underlined):

MGAPRSLLLALAAGLAVARPPNIVLIFADDLGYGDLGCYGHPSSTTPNLDQLAAGGLRFTDFYVPVSLCTPSRAALLTGRLPVRMGMYPGVLVPSSRGGLPLEEVTVAEVLAARGYLTGMAGKWHLGVGPEGAFLPPHQGFHRFLGIPYSHDQGPCQNLTCFPPATPCDGGCDQGLVPIPLLANLSVEAQPPWLPGLEAR Y MAFAHDLMADAQRQDRPFFLYYASHHTHYPQFSGQSFAERSGRGPFGDSLMELDAAVGTLMTAIGDLGLLEETLVIFTADNGPE T MRMS R GGCSGLLRCGKGTTYEGGVREPALAFWPGHIAPGVTHELASSLDLLPTLAALAGAPLPNVTLDGFDLSPLLLGTGKSPRQSLFFYPSYPDEVRGVFAVRTGKYKAHFFTQGSAHSDTTADPACHASSSLTAHEPPLLYDLSKDPGENYNLLGGVAGATPEVLQALKQLQLLKAQLDAAVTFGPSQVARGEDPALQICCHPGCTPRPACC HCPDPHA

SEQ ID NO: 2 shows the wild type human ARSA encoding nucleic acidsequence (cDNA). The preferred positions for mutations are in bold andunderlined.

GGGGCACCGCGGTCCCTCCTCCTGGCCCTGGCTGCTGGCCTGGCCGTTGCCCGTCCGCCCAACATCGTGCTGATCYTTGCCGACGACCTCGGCTATGGGGACCTGGGCTGCTATGGGCACCCCAGCTCTACCACTCCCAACCTGGACCAGCTGGCGGCGGGAGGGCTGCGGTTCACAGACTTCTACGTGCCTGTGTCTCTGTGCACACCCTCTAGGGCCGCCCTCCTGACCGGCCGGCTCCCGGTTCGGATGGGCATGTACCCTGGCGTCCTGGTGCCCAGCTCCCGGGGGGGCCTGCCCCTGGAGGAGGTGACCGTGGCCGAAGTCCTGGCTGCCCGAGGCTACCTCACAGGAATGGCCGGCAAGTGGCACCTTGGGGTGGGGCCTGAGGGGGCCTTCCTGCCCCCCCATCAGGGCTTCCATCGATTTCTAGGCATCCCGTACTCCCACGACCAGGGCCCCTGCCAGAACCTGACCTGCTTCCCGCCGGCCACTCCTTGCGACGGTGGCTGTGACCAGGGCCTGGTCCCCATCCCACTGTTGGCCAACCTGTCCGTGGAGGCGCAGCCCCCCTGGCTGCCCGGACTAGAGGCCCG CTAC ATGGCTTTCGCCCATGACCTCATGGCCGACGCCCAGCGCCAGGATCGCCCCTTCTTCCTGTACTATGCCTCTCACCACACCCACTACCCTCAGTTCAGTGGGCAGAGCTTTGCAGAGCGTTCAGGCCGCGGGCCATTTGGGGACTCCCTGATGGAGCTGGATGCAGCTGTGGGGACCCTGATGACAGCCATAGGGGACCTGGGGCTGCTTGAAGAGACGCTGGTCATCTTCACTGCAGACAATGGA CCTGAG ACCATGCGTATGTCC CGA GGCGGCTGCTCCGGTCTCTTGCGGTGTGGAAAGGGAACGACCTACGAGGGCGGTGTCCGAGAGCCTGCCTTGGCCTTCTGGCCAGGTCATATCGCTCCCGGCGTGACCCACGAGCTGGCCAGCTCCCTGGACCTGCTGCCTACCCTGGCAGCCCTGGCTGGGGCCCCACTGCCCAATGTCACCTTGGATGGCTTTGACCTCAGCCCCCTGCTGCTGGGCACAGGCAAGAGCCCTCGGCAGTCTCTCTTCTTCTACCCGTCCTACCCAGACGAGGTCCGTGGGGTTTTTGCTGTGCGGACTGGAAAGTACAAGGCTCACTTCTTCACCCAGGGCTCTGCCCACAGTGATACCACTGCAGACCCTGCCTGCCACGCCTCCAGCTCTCTGACTGCTCATGAGCCCCCGCTGCTCTATGACCTGTCCAAGGACCCTGGTGAGAACTACAACCTGCTGGGGGGTGTGGCCGGGGCCACCCCAGAGGTGCTGCAAGCCCTGAAACAGCTTCAGCTGCTCAAGGCCCAGTTAGACGCAGCTGTGACCTTCGGCCCCAGCCAGGTGGCCCGGGGCGAGGACCCCGCCCTGCAGATCTGCTGTCATCCTGGCTGCACCCCCCGCCCAGCTTGCTGCCATTGCCCAGATCCCCATGCC

SEQ ID NO: 3 shows the amino acid sequence of a mutated ARSA of theinvention including one amino acid substitution. The mutation is boldand underlined.

MGAPRSLLLALAAGLAVARPPNIVLIFADDLGYGDLGCYGHPSSTTPNLDQLAAGGLRFTDFYVPVSLCTPSRAALLTGRLPVRMGMYPGVLVPSSRGGLPLEEVTVAEVLAARGYLTGMAGKWHLGVGPEGAFLPPHQGFHRFLGIPYSHDQGPCQNLTCFPPATPCDGGCDQGLVPIPLLANLSVEAQPPWLPGLEAR Y VAFAHDLMADAQRQDRPFFLYYASHHTHYPQFSGQSFAERSGRGPFGDSLMELDAAVGTLMTAIGDLGLLEETLVIFTADNGPETMRMSRGGCSGLLRCGKGTTYEGGVREPALAFWPGHIAPGVTHELASSLDLLPTLAALAGAPLPNVTLDGFDLSPLLLGTGKSPRQSLFFYPSYPDEVRGVFAVRTGKYKAHFFTQGSAHSDTTADPACHASSSLTAHEPPLLYDLSKDPGENYNLLGGVAGATPEVLQALKQLQLLKAQLDAAVTFGPSQVARGEDPALQICCHPGCTPRPACC HCPDPHA

SEQ ID NO: 4 shows the amino acid sequence of a mutated ARSA of theinvention including three amino acid substitution. The mutations arebold and underlined.

MGAPRSLLLALAAGLAVARPPNIVLIFADDLGYGDLGCYGHPSSTTPNLDQLAAGGLRFTDFYVPVSLCTPSRAALLTGRLPVRMGMYPGVLVPSSRGGLPLEEVTVAEVLAARGYLTGMAGKWHLGVGPEGAFLPPHQGFHRFLGIPYSHDQGPCQNLTCFPPATPCDGGCDQGLVPIPLLANLSVEAQPPWLPGLEAR Y VAFAHDLMADAQRQDRPFFLYYASHHTHYPQFSGQSFAERSGRGPFGDSLMELDAAVGTLMTAIGDLGLLEETLVIFTADNGPE L MRMS N GGCSGLLRCGKGTTYEGGVREPALAFWPGHIAPGVTHELASSLDLLPTLAALAGAPLPNVTLDGFDLSPLLLGTGKSPRQSLFFYPSYPDEVRGVFAVRTGKYKAHFFTQGSAHSDTTADPACHASSSLTAHEPPLLYDLSKDPGENYNLLGGVAGATPEVLQALKQLQLLKAQLDAAVTFGPSQVARGEDPALQICCHPGCTPRPACC HCPDPHA

EXAMPLES Example 1 Comparison of Human and Murine ARSA Enzyme Activity

The rate of galactosylceramide (galcer) formation was measured by anestablished micellar assay (Matzner U et al., J Biol Chem, 2009, 284,9372-81). For the reaction, purified ARSA (ASA, 1 μg) was incubated with5 nmol sulfatide (sulf) in the presence of 0.33 nmol saposin B (SapB) in10 mM sodium acetate buffer pH 4.5 at 37° C. Experiments were done intriplicates (#1-3). After incubation times of 30 and 60 min,respectively, lipids were extracted (Folch J et al., J Biol Chem., 1957,226, 497-509) and separated by thin layer chromatography. Under in vitroconditions, the bile salt taurodeoxycholate (TDC), but not unconjugateddeoxycholate, can functionally substitute for SapB. TDC (100 nmol) anddeoxycholate (100 nmol) were used instead of SapB in positive (pos) andnegative controls (neg), respectively. Results are shown in FIG. 1.Additional negative controls contained 1 μg bovine serum albumin (BSA)instead of ARSA. An equimolar mixture of sulfatide andgalactosylceramide (sulf/galcer 1:1) was used as a lipid standard.

The intensity of the galactosylceramide band is a measure for thecatalytic rate of ARSA. The densitometric evaluation of thegalactosylceramide band (not shown) revealed a 3- to 4-fold highercatalytic rate of murine ARSA compared to human ARSA.

Example 2 Mutagenesis of Human ARSA

In order to identify targets responsible for the increased activity ofmurine ARSA compared to human ARSA, the amino acid sequences of bothenzymes were compared (FIG. 2). Amino acid substitutions tend to occurin clusters defining a mosaic of nine variable and eight constantdomains. These are highlighted by bold orange numbers 1-9 (whitebackground) and bold green numbers 1-8 (green background), respectively.Four unclustered amino acid exchanges which are located in constantregions are designated as “human-specific modifications” (hsm's) and arenumbered from hsm-1 to hsm-4 (vertical captions in orange). Legend: bluebox—signal peptide; red box—alpha helix; red arrow—beta sheat;underlined—surface localization; bold green—important for active sitegeometry; blue amino acids—conservative exchange (+), red aminoacids—non-conservative exchange; red (vertical captions)—amino acidexchanges leading to severe MLD; black (vertical)—MLD with unknownseverity; green (vertical)—mild MLD; blue (vertical)—polymorphism.

Using site-directed mutagenesis (see FIG. 3), the variable domains v1 tov9 and the human-specific modifications hsm1 to hsm4 of the human ARSA(dark grey) were exchanged by homologous sequences from the murine ARSA(light grey). The resulting man-mouse chimeric ARSAs were analysed byactivity assays as described in FIGS. 4 and 5.

In FIG. 4, left-hand side, a Strep-tag was fused to the N-terminus ofthe full length human ARSA cDNA and the coding sequence of theStrep-tagged ARSA was inserted into the eukaryotic expression plasmidpcDNA3. Amino acids of the parental construct pcDNA3-ASA-Strep weresubstituted by their murine homologues using site-directed mutagenesisas indicated.

In FIG. 4, right-hand side: To measure the activity of the murinizedARSA polypeptides, chinese hamster ovary-K1 cells (CHO-K1) weretransfected with the mutated expression plasmids pcDNA3-ASA-Strepmut.Binding of the overexpressed ARSA polypeptides to the mannose6-phosphate receptors was inhibited by addition of 10 mM ammoniumchloride. This resulted in the bulk secretion of the newly synthesizedlysosomal enzymes and allowed analysis of the murinized ARSAs in theconditioned media. The activity and concentration of the secreted ARSAwas measured with the artificial substrate para-nitrocatechol sulfate(Baum H et al., Clin Chim Acta. 1959, 4, 453-455) and a sensitivesandwich ELISA being specific for the human ARSA (Matzner U et al., GeneTher. 2000, 7, 805-12). To determine the background activity ofendogenous hamster-ARSA in the medium, CHO-K1 control cells weretransfected with pcDNA3 (empty vector). The specific activity of mutatedARSA (mU/μg) was calculated by subtracting this background activity anddividing the result (mU/ml) through the ARSA concentration (μg/ml).

As shown in FIG. 5, the variable domains v1 to v9 of the human ARSAamino acid sequence (dark grey) were individually exchanged byhomologous sequences from the murine ARSA (light grey). The murinizedARSA polypeptides and wild-type ARSA (hASA) were expressed in CHO-K1cells and their specific activity was determined as described for FIG.4—results are provided in FIG. 5. Bars represent means±SDs of theindicated number of independent transfection experiments (n=4-22). Astatistically significant difference to the wild-type ARSA is indicatedby an asterisc (Student's t-test). For respective P-values and foldincreases to wild-type ARSA see FIG. 5C. The murinization of the“human-specific modifications” hsm1 to hsm4 (see FIG. 3) had nosignificant effect on the specific activity of the human ARSA (notshown).

Based on the observation that murinization of either v4 or v6 increasedthe specific activity of the human ARSA (see FIG. 5), these two and theinterjacent variable domain v5 were exchanged by murine sequences (lightgrey) in the indicated combinations (FIG. 6A). The specific activitiesof ARSAs with a combined exchange of v4 and v6 or a combined exchange ofv4, v5 and v6 are higher than those with single exchanges of v4 and v6(FIG. 6B). For P-values (Student's t-test) and fold differences towild-type ARSA see FIG. 6C.

Various combinations of amino acid and domain exchanges were constructedto identify individual amino acids in v4 and v6 that increase the ARSAactivity (FIG. 7A). For each murinized position the human amino acid(dark grey), position (black) and murine amino acid (light grey) isshown. A blue box indicates that the entire variable domain wasmurinized. A combined exchange of human M202 (to murine V202) and humanv6 (to murine v6) has the greatest effect and increases the meanspecific activity 5.4-fold compared to wild-type human ARSA (FIG. 7B).The difference is statistically significant (Student's t-test;P=6.6×10−8, n=9 and 22, respectively).

The construct M202V, x:v6 combines the three amino acid exchanges M202V,T286L and R291N (FIG. 8). To possibly detect combinations with evenhigher specific activity, M202V was combined with a variety ofindividual amino acid exchanges in v4 and v6. Exchanges in brackets areconservative. None of the tested combinations was superior to M202V,x:v6 (=M202V, T286L, R291N). Bars represent means±SDs of the indicatednumber of n=4-22 independent transfection experiments.

To detect amino acid exchanges in variable domain v5 which mightincrease the specific activity of M202V, T286L, R291N (=M202V, x:v6)individual amino acids of v5 (T260, 1265) or the entire v5-domain wasmurinized as indicated (FIG. 9). None of the exchanges increased thespecific activity compared to M202V, T286L, R291N. Bars representmeans±SDs of n=4-22 independent transfection experiments.

Example 3 Specific Activities of ARSA Mutants

To determine the specific activities of the murinized ARSA polypeptidesfour different methods to measure enzyme concentrations were compared(FIG. 10). The tables indicate the specific activities in mU/μg (firstcolumn) and fold increase compared to wild-type ARSA (second column).Human and murine ARSA is abbreviated as hASA and mASA, respectively.Sandwich ELISA of conditoned media using Strep-Tactin to immobilize ARSAvia its Strep-tag. A polyclonal anti-human ARSA antiserum was used assecondary antibody (FIG. 10A). Sandwich ELISA using a monoclonalantibody specific for human ARSA as a capture antibody. A polyclonalrabbit anti-human ARSA antiserum was used for detection (FIG. 10B).Silver staining of ARSA polypeptides purified from the conditioned mediaof transfected CHO-K1 cells via Strep-Tactin affinity chromatography(FIG. 10C). Western blotting of ARSA polypeptides purified fromconditioned media of transfected CHO-K1 cells via Strep-Tactin affinitychromatography (FIG. 10D). Peroxidase-conjugated Strep-Tactin was usedto visualize the ARSA polypeptides. Depending on the quantificationmethod and the source of enzyme (purified or unpurified) the ARSA mutantM202V,T286L,R291N shows a 5.5 to 2.1-fold increase of specific activitycompared to wild-type ARSA.

Example 4 Endocytosis of Mutated ARSA

In preparation of a proof-of-concept study demonstrating increasedtherapeutic efficacy of the hyperactive ARSA mutants additionalexperiments were conducted. In particular, the endocytosis, stabilityand immunogenicity of the hyperactive ARSA mutants were analysed.Furthermore, the recombinant ARSAs were purified in milligram amountsbeing sufficient for a preclinical enzyme replacement trial in the nearfuture. For this purpose, ARSA_M202V and ARSA_M202V,T286L,R291N werecontinuously expressed over 6 months as Strep-tagged recombinantproteins by Chinese hamster ovary (CHO) suspension cells and isolatedfrom the conditioned medium by affinity chromatography. In parallel,similar amounts of Strep-tagged wildtype human ARSA and Strep-taggedwildtype murine ARSA were purified as controls.

Enzyme replacement therapy depends on efficient uptake of the infusedlysosomal enzyme by the enzyme-deficient cells of the patient. ARSA isprimarily endocytosed via mannose 6-phosphate receptors that recognizemannose 6-phosphate residues that are attached to the N-glycans of theenzyme during its synthesis in the endoplasmic reticulum and Golgiapparatus. To analyse a possible adverse effect of the mutations on thisposttranslational modification and the endocytic rate, CHO-K1 cells werefed with recombinantly expressed ARSA mutants or wildtype human ARSA for24 h and the amount of internalized ARSA was determined by activitymeasurements. No significant difference in the endocytosis of wildtypehuman ARSA, ARSA_M202V and ARSA_M202V,T286L,R291N was discernible (FIG.11). The uptake rates were comparable to that of industriallymanufactured (and efficiently phosphoryated) human ARSA used in currentclinical trials. This suggests a normal mannose 6-phosphorylation of theARSA mutants.

Example 5 Stability of Mutated ARSA

Higher enzymatic activity can be a consequence of increasedconformational flexibility of loop and hinge regions in the polypeptidescaffold promoting the active site dynamics and the velocity of thecatalytic cycle. The stability of an enzyme is therefore often inverselycorrelated with its activity (Miller, S R.; 2017 Evolution 71,1876-1887). To analyse possible consequences of the activity-promotingamino acid exchanges M202V,T286L and R291N, the stability of the fourrecombinantly expressed ARSAs in solution (shelf life) and within cells(lysosomal half life) was analysed. Storage in Tris-buffered saline pH7.4 at 4° C. for up to 10 days diminished the enzyme activities of therecombinant ARSAs by approximately 10% with no clear difference betweenthe four preparations (FIG. 12A). Likewise, repeated freeze-thaw cyclesreduced the activities of all four ARSAs slightly and to a similarextent (FIG. 12B). Thus, the mutations did not significantly affect theshelf life of the enzyme. In contrast, clear differences between theARSA preparations were discernible when their intralysosomal stabilitieswere determined (FIG. 12C). Pulse feeding experiments revealed halflives of 62 h, 57 h, 46 h and 39 h for wildtype human ARSA, ARSA_M202V,ARSA_M202V,T286L,R291N and wildtype murine ARSA, respectively. Thus, thesingle mutation M202V and the triple mutation M202V,T286L,R291N diminishin fact the stability of the human ARSA in its normal subcellularenvironment indicating an inverse correlation between activity andstability. It has to be emphasized, however, that the factor of activityincrease (3.4-fold and 5.4-fold, respectively) outweighs by far thisloss of stability (8% and 26%, respectively). This can be concluded fromthe following pharmakinetic considerations: When lysosomal ARSA activityis plotted versus time after dosage, the integral or “area under thecurve” is a measure for the bioavailability of ARSA and its potency todegrade sulfatide storage. Taking into account identical endocytic rates(FIG. 11), a mono-exponential decline of lysosomal concentrations (FIG.12C) and the experimentally determined half lives and factors ofactivity increase, the areas under the curves are 3.1- and 4.0-foldlarger for ARSA_M202V and ARSA_M202V,T286L,R291N compared to wildtypehuman ARSA (calculation not shown). Thus, the observed decline instability will only slightly restrict the increased potency of thehyperactive ARSA mutants to hydrolyse sulfatide.

Example 6 Immunogenicity of Mutated ARSA

To analyse possible new epitopes and immunogenicities introduced intothe human ARSA polypeptide by the amino acid exchanges, an MLD mousemodel was treated by repeated intravenous injections of wildtype humanARSA, ARSA_M202V and ARSA_M202V,T286L,R291N, respectively. Treatmentswere done in weekly intervals for a total of four weeks (fourinjections) using 20 mg enzyme per kg body weight in each injection. TheARSA knockout mouse model used for this study was transgenic for anactive sitemutant of the human ARSA. This ARSA variant has zero activityand has been constructed by an amino acid exchange in the substratebinding pocket that does not affect the surface structure of the enzyme(Matzner, U., et al. Mol. Med. 13, 471-479; 2007). Consequently, themouse strain retains its MLD-like phenotype, but does not develop immunereactions to injected wildtype human ARSA. ARSA knockout mice withoutthis transgene show, in contrast, deteriorating adverse reactions withthe second injection and more than 50% have died from anaphylacticcomplications after the fourth injection of 20 mg/kg wildtype humanARSA. By this means, repeated treatment of the immunotolerant mousestrain allows conclusions about possible new immunogenicities of thehuman ARSA mutants.

Treatment of the immunotolerant mouse model with either wildtype humanARSA, ARSA_M202V or ARSA_M202V,T286L,R291N for four weeks caused noobvious behavioral side effects (n=3 mice per group). Treatment with themurine ARSA, on the contrary, elicited apparent incompatibilityreactions such as bristling of the fur, unsteady gait and reduced cageactivity. These reactions were transient and occurred 5 to 20 min aftertreatment in two of the three mice. Signs were observed for the firsttime after the third and were more pronounced after the fourthinjection. The third mouse treated with mARSA showed no behavioralabnormalities except enhanced skin scratching 5 to 10 min aftertreatment possibly related to histamine-induced itch.

To analyse the development of antibodies to repeatedly infused ARSA,blood was taken three days after the fourth treatment. Antibody titerswere measured by the capability of serum to precipitate that recombinantARSA from solution that had been used for treatment (Matzner, U et al.(2008) J. Mol. Med. (Berl.) 86, 433-442). In this assay, the amount ofARSA lost from the supernatant is a measure for the α-ARSA antibodyconcentration. Serum from the three mice that had received wildtypehuman ARSA did not precipitate human ARSA from solution indicating theabsence of antibodies and confirming the immunotolerance of the mice(FIG. 13A). Likewise, none of the ARSA_M202V and ARSA_M202V,T286L,R291Ntreated mice showed antibodies to the ARSA-variant used for treatment(FIG. 13B). Among the three mice treated with murine ARSA, on thecontrary, one exhibited a high concentration of antibodies to murineARSA.

The behavioral and biochemical data indicate that expression of wildtypehuman ARSA fully protects from immune reactions to ARSA_M202V andARSA_M202V,T286L,R291N, but only partially to adverse reactions tomurine ARSA. Though the mice respond not equally to murine ARSA in thisshort treatment period of four weeks, it is likely, that they willdevelop a progressive immune response in the long range. It has to bementioned that approximately 94% of European MLD patients express humanARSA polypeptides, though at a decreased level or with markedly reducedactivity (Polten, A et al (1991). N. Engl. J. Med. 324, 18-22). Thepreclinical data presented here suggest that ARSA_M202V andARSA_M202V,T286L,R291N will not cause immunological complications inthis majority of patients.

Materials and Methods

Purification of Recombinant ARSAs

For the production of recombinant proteins, CHO-suspension (CHO-S) cells(Thermo Fisher Scientific) were stably transfected withpcDNA3-hARSA-strep, pcDNA3-mARSA-strep, pcDNA3-hARSA_M202V-strep andpcDNA3-hARSA_M202V,T286L,R291N-strep, respectively. Transfection,selection, isolation and screening of single clones as well asproduction of recombinant ARSA was as described before³. Briefly, mediumwas collected twice a week from Miniperm bioreactors (Sarstedt,Nürnbrecht, Germany) and mixed with 50% (w/v) ammonium sulfate toprecipitate ARSA. Precipitates were stored at 4° C. For affinitypurification, the precipitated ARSAs were collected by centrifugation(1,500×g, 4° C., 30 min) and then excessively dialysed againstTris-buffered saline pH 7.4 at 4° C. Insoluble material was removed bycentrifugation (100,000×g, 4° C., 60 min) and recombinant ARSA wassubsequently purified from the supernatant by affinity chromatographyusing Strep-Tactin Macroprep® (IBA Lifesciences, Göttingen, Germany)according to the manufacturers recommendations.

Endocytosis and Stability

To determine the endocytic rate of recombinant ARSAs, CHO-K1 cells werecultured for 24 h in cell culture medium to which the respectiverecombinant ARSA was added at a concentration of 2.5 μg/ml. Then thecells were washed with 1× PBS pH 7.4 and cultured in fresh medium fordifferent chase times. Before harvesting, cells were washed for 3 min atroom temperature with 50 mM Glycin, 150 mM NaCl, pH 3.0 to removesurface-bound ARSA. Following trypsinization, cells were spun down andhomogenized in 100 μl homogenization buffer (0.5% Triton N-101 in 1× TBSpH 7.0). For endocytosis experiments, cells were harvested immediatelyafter feeding and the ARSA activity of the homogenate was measured.Activities were corrected by subtracting the activitity of CHO-K1 cellscultured without recombinant ARSAs (mean of n=3 dishes) and related tothe activity of the incubation medium added to the cells at t₀. Thelysosomal stability was analysed by Western blotting. For that purpose,aliquots of homogenates (20μl) or incubation media (4 μl) were separatedby SDS-PAGE. ARSA was detected with a mixture of the two polyclonalrabbit antisera #1057 (specific for human ARSA, 1:10.000) and N14 (SantaCruz Biotechnology, Heidelberg, Germany; sc-79848; detects also murineARSA; 1:200). The antisera were used in combination withperoxidase-conjugated goat-anti-rabbit (Dianova, Hamburg, Germany;111-035-003; 1:10.000) as secondary antibody. ARSAs were quantified bydensitometry of signals using the image analysis software AIDA (Raytest,Straubenhardt, Germany). Time course data were fitted to themono-exponential equation N(t)=N₀ e^(−λt), using the least square method(Microsoft Excel 2010). Half-lives were calculated according to theformula T_(1/2)=(1n2)/λ.

Tolerability Study

ARSA knockout mice being immunotolerant to wildtype human ARSA (Baum, H.et al 1959 Clin. Chim. Acta 4, 453-455.) were treated by repeatedintravenous injection of high doses of recombinant ARSAs into the tailvein. For this purpose, four groups of age- and sex-matchedimmunotolerant ARSA knockout mice (13 months old females, n=3 mice pergroup) were injected with one recombinant ARSA preparation each using atreatment dose of 20 mg per kg body weight given once a week for a totalof four weeks (four injections). A fifth group of mice was mock-treatedwith buffer (1× TBS pH 7.4) according to the same schedule. Acute immunecomplications such as scratching, wiping of eyes, bristling of the furand reduced cage activity were analysed by visual inspection of the micewithin the first 30 min after each injection. The formation ofantibodies was determined by the ability of serum isolated three daysafter the fourth treatment to immunoprecipitate the ARSA that has beenused for treatment from solution (Matzner, U., et al (2008) J. Mol. Med.(Berl.) 86, 433-442.).

1. A mutated arylsulfatase A (ARSA) enzyme, or a functional fragmentthereof, comprising an amino acid sequence with at least 80% sequenceidentity to SEQ ID NO: 1, wherein the mutated ARSA enzyme amino acidsequence when aligned to the sequence of SEQ ID NO: 1, comprises atleast one mutation compared to the sequence between residues 100 and 400of SEQ ID NO:
 1. 2. The mutated ARSA enzyme, or the functional fragmentthereof, according to claim 1, wherein the amino acid sequence of themutated ARSA enzyme, or of the functional fragment thereof, when alignedto the sequence of SEQ ID NO: 1, comprises at least one mutation atamino acid positions 202, 286 and/or 291 of SEQ ID NO: 1, and,preferably, comprises an amino acid sequence at least 80% identical tothe sequence of SEQ ID NO: 3 or
 4. 3. The mutated ARSA enzyme, or thefunctional fragment thereof, according to claim 1 or 2, wherein themutation is selected from a substitution, deletion, addition, insertionor amino acid modification, and preferably is an amino acidsubstitution.
 4. The mutated ARSA enzyme, or the functional fragmentthereof, according to any one of claims 1 to 3, wherein the mutation isa murinization of a residue in the human ARSA enzyme to a correspondingmurine ARSA enzyme residue.
 5. The mutated ARSA enzyme, or thefunctional fragment thereof, according to any one of claims 1 to 4,wherein the amino acid sequence of the mutated ARSA enzyme, or of thefunctional fragment thereof, when aligned to the sequence of SEQ ID NO:1, comprises at least one mutation selected from M202V, T286L and/orR291N compared to SEQ ID NO: 1, preferably of at least M202V.
 6. Themutated ARSA enzyme, or the functional fragment thereof, according toany one of claims 1 to 5, wherein mutated ARSA enzyme, or the functionalfragment thereof, retains an enzymatic activity of degradation ofsulfatides, preferably an activity of degradation of cerebroside3-sulfate into cerebroside and sulfate.
 7. An isolated nucleic acidcomprising a sequence coding for the mutated ARSA enzyme, or thefunctional fragment thereof, according to any one of claims 1 to
 6. 8. Avector, comprising the nucleic acid according to claim
 7. 9. The vectoraccording to claim 8, which is an expression vector, comprising promotersequence operably linked to the nucleic acid according to claim
 7. 10. Arecombinant cell comprising a mutated ARSA enzyme, or the functionalfragment thereof, according to any one of claims 1 to 6, a nucleic acidaccording to claim 7, or a vector according to any one of claims 8 or 9.11. The recombinant cell according to claim 16, which is a bacterialcell, an insect cell or a vertebrate, preferably a mammalian cell suchas a Chinese Hamster Ovary (CHO) cell, or a hematopoietic stem cell(HSC).
 12. A pharmaceutical composition comprising the mutated ARSAenzyme, or the functional fragment thereof, according to any one ofclaims 1 to 6, or a nucleic acid according to claim 7, or a vectoraccording to claim 8 or 9, or a recombinant cell according to claim 10or 11, together with a pharmaceutically acceptable carrier, stabilizerand/or excipient.
 13. A compound for use in the treatment of a disease,the compound being selected from a mutated ARSA enzyme, or thefunctional fragment thereof, according to any one of claims 1 to 6, or anucleic acid according to claim 7, or a vector according to claim 8 or9, or a recombinant cell according to claim 10 or 11, or apharmaceutical composition according to claim
 12. 14. A method forproducing the mutated ARSA enzyme, or the functional fragment thereof,according to any one of claims 1 to 6, or a nucleic acid according toclaim 7, or a vector according to claim 8 or 9, or a recombinant cellaccording to claim 10 or 11, or a pharmaceutical composition accordingto claim
 12. 15. A method for designing and/or producing a mutated ARSAenzyme, or a functional fragment thereof, comprising the steps of (a)providing a parent ARSA enzyme-encoding nucleic acid sequence whichencodes a parent ARSA enzyme having an amino acid sequence with at least80% sequence identity to SEQ ID NO: 1, or a functional fragment thereof,(b) introducing into said parent ARSA enzyme-encoding nucleic acidsequence, or the functional fragment thereof, at least one mutation,thereby generating a mutated ARSA enzyme-, or functional fragmentthereof, -encoding nucleic acid sequence, wherein the mutated ARSAenzyme, or functional fragment thereof, -encoding nucleic acid sequenceencodes a mutated ARSA enzyme, or a functional fragment thereof,com-comprising a mutated ARSA enzyme amino acid sequence, that, whenaligned to the sequence of SEQ ID NO: 1, comprises at least one mutatedamino acid residue compared to the sequence of SEQ ID NO: 1 betweenresidues 100 and 400, and wherein said at least one mutated amino acidresidue constitutes a mutation when compared to the amino acid sequenceof the parent ARSA enzyme, (c) Optionally, expressing said mutated ARSAenzyme-, or functional fragment thereof, -encoding nucleic acidsequence, to obtain a mutated ARSA enzyme, or the functional fragmentthereof.